There is an urgent need to develop more effective diagnostic and therapeutic strategies for cancer. For example, conventional therapies, such as chemo- and/or radio-therapy, are often unable to eradicate cancers because of tumor specificities, drug/radiation resistance, and significant side effects.
Given their roles as important regulators of gene expression and genome integrity in cellular development, physiology and pathology, small non-coding RNAs (ncRNAs) have great therapeutic potential for the treatment of cancer. Current translational research of ncRNA-based therapeutics focuses on small interfering RNA (siRNA) and microRNA (miRNA)-based approaches; these are now being tested in clinical trials. Their high efficacy, target-specific action and low toxicity offer significant advantages over the current conventional treatments of cancer. Historically, there are six types of NA-based products: antisense, ribonucleic acid inhibition (RNAi), gene therapy, nucleoside analogs, ribozymes, and aptamers.
PIWI-interacting RNAs (piRNAs) are small (mostly 26-32 nt) noncoding RNAs with highly conserved functions in the protection of germline stem cells from transposable element mobilization. Like microRNAs and small interfering RNAs, piRNAs act as guides in sequence-specific gene regulation in conjunction with Argonaute proteins (PIWI, rather than AGO sub-family proteins, FIG. 1A), yet are far more abundant-over 30,000 piRNAs have been identified in humans, and this number is likely far greater as millions have been identified in other mammalian cells. PIWI-piRNA ribonucleoprotein complexes recruit chromatin-remodeling machinery to complementary genomic targets, where heritable epigenetic modifications are established (via DNA methylation in mammals). Recent studies have also proposed that piRNAs may act post-transcriptionally in mRNA silencing.
Mobile genetic elements such as transposons are a constant threat for the genome. PIWI-interacting RNAs (piRNAs) protect germline cells from transposons in organisms as diverse as flies, fish and mammals. piRNAs are 25 to 33 nt in length, depending on the PIWI clade protein that they bind to. piRNAs derive from distinct transposons that are referred to as piRNA clusters, but the piRNAs from each locus are characterized by a complex mixture of sequences spanning large portions of the transposon. piRNA clusters are transcribed in the sense or antisense direction, and the long single-stranded RNA serves as the basis for piRNA production.
The biogenesis of piRNAs is independent of Dicer and requires other nucleases. Two biogenesis pathways are important for piRNA production. First, a primary processing pathway generates primary piRNAs, and these are then amplified by an amplification cycle referred to as the ping-pong loop. In the primary biogenesis pathway, the long transposon transcript is initially cleaved by the nuclease zucchini (FIGS. 1A and 1B), which probably generates the 5′ ends of primary piRNAs.
In the ping-pong cycle (see the lower right panel of FIG. 1B), mature sense primary piRNAs guide PIWI clade proteins to complementary sequences on antisense transcripts from the same piRNA cluster. PIWI proteins use their slicer activity to cleave the target antisense transcript to generate a new 5′ end. This 5′ end is bound by another PIWI protein. In subsequent steps, the 3′ end is trimmed to the length of the mature piRNA, leading to a mature antisense secondary piRNA, which can now target sense transcripts transcribed from the piRNA cluster. In Drosophila melanogaster, the two PIWI proteins Aubergine and Ago3 cooperate in secondary piRNA production to generate sense and antisense piRNAs. However, antisense piRNAs dominate, and a protein called Qin, which contains E3 ligase and Tudor domains, seems to modulate such a heterotypic ping-pong cycle. In the mouse germ line, the PIWI proteins MILI and MIWI collaborate in piRNA generation. After trimming, piRNAs receive a methyl group at the 3′ end by the methyltransferase HEN1. Primary piRNAs carry such modifications as well.
piRNAs guide PIWI proteins to complementary RNAs derived from transposable elements. Similar to RNA interference, PIWI proteins cleave the transposon RNA, leading to silencing. In flies, mutations in piwi, aub and Ago3 (which encode the PIWI proteins in D. melanogaster) are required for transposon silencing in the germ line. Similar observations were made when the mouse PIWI proteins MILI and MIWI were genetically inactivated. Here, long interspersed nuclear elements (LINE) and long terminal repeat (LTR) retrotransposons accumulated.
Despite the longstanding notion that activity of the PIWI-piRNA pathway is restricted to the germline, evidence is quickly mounting for roles in somatic tissues, particularly in the context of cancer. Aberrant PIWI-family protein expression has been associated with unfavorable prognosis in eleven cancer types, and piRNA expression has been observed in fourteen cancer types. In the most comprehensive study of piRNA expression outside of the germline to date, Martinez et al. utilized RNA-seq data from The Cancer Genome Atlas to demonstrate that hundreds of piRNAs are expressed in both normal and malignant tissues from each of eleven anatomical sites (bladder, breast, colon, head/neck, kidney, lung, ovaries, prostate, stomach, thyroid, and uterus), and that piRNA expression programs are dysregulated in a clinically relevant, tumor type-specific manner.
Studies show that aberrant piRNA expression can be a signature feature of a cancer. However, over 20,000 piRNA genes in the human genome and irregular piRNA expression appears to be cancer type specific. Thus, there remains a need to identify the piRNA's whose aberrant expression correlates with a particular cancer's frequency and/or severity, and design therapeutic measure based thereon.
Thus, it is an object of the invention to provide specific piRNAs thereof that are genetically associated or are aberrantly expressed in specific cancer types including glioblastoma, liver, prostate, lung, and breast.
It is another object of the invention to provide therapeutic agents and methods of use thereof to correct or compensate for aberrant piRNA expression in a subject in need thereof.
It is another object of the invention to provide methods of diagnosing a subject or predicting the severity of the subject's disease.
It is another object of the invention to provide methods of determining the efficacy of a therapeutic intervention.
It is another object of the invention to provide methods of screening for new aberrant piRNA.